Bottom Line:
There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts.However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically.Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

Affiliation: Center for Cardiovascular and Muscle Research, Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 112031, USA.

ABSTRACTIn order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP-1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP-1 +/- mice hearts. Using the isolated heart model, we observed that the CLP-1 +/- hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP-1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild-type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts. However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK-1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

f5: Expression of PGC‐1α and HIF‐1α in heart‐tissue of CLP‐1 heterozygous mice. (A) The expression levels of PGC‐1α in the protein heart extracts from control, preconditioning (PC), ischemia/reperfusion (I/R) and I/R with PC groups of wild‐type and in CLP‐1 +/− hearts was determined by Western blot analysis. GAPDH was used as loading control. (B) We use the same extracts as in Figure 4A to determine the expression levels of HIF‐1α, and as before GAPDH was used as loading control. The experiment was repeated three times and a representative experiment is shown. *P < 0.05 versus wild‐type control, **P < 0.01 versus wild‐type control. (C) The ubiquitination profile of HIF‐1 was examined by using heart extracts from each group. The extracts were used for immunoprecipitation with anti‐ubiquitin antibody followed by Western blot with an antibody against HIF‐1α. (D) The interaction between HIF‐1α and SUMO‐1 was determined by immunoprecipitations using anti− HIF‐1α antibody followed by Western blotting with anti‐SUMO‐1 antibody. Western blot with anti− HIF‐1α antibody served as loading control. (E) Western blot was performed as in (A) to evaluate expression of SUMO‐1. GAPDH was used as loading control. (F) Expression of pyruvate dehydrogenase kinase‐1 (PDK‐1) was evaluated by Western blot using antibody against PDK‐1. The representative figure shows increase in PDK‐1 expression in extracts from CLP‐1 +/− mice hearts subjected to stress. GAPDH expression was used as loading control.

Mentions:
(A) Schematic representation of the experimental protocol used for the evaluation of haemodynamic parameters and infarct size. (B) A diagram of the experimental protocols for stress application and analysis of wild‐type and CLP‐1 +/− hearts (see Figs. 4 and 5).

f5: Expression of PGC‐1α and HIF‐1α in heart‐tissue of CLP‐1 heterozygous mice. (A) The expression levels of PGC‐1α in the protein heart extracts from control, preconditioning (PC), ischemia/reperfusion (I/R) and I/R with PC groups of wild‐type and in CLP‐1 +/− hearts was determined by Western blot analysis. GAPDH was used as loading control. (B) We use the same extracts as in Figure 4A to determine the expression levels of HIF‐1α, and as before GAPDH was used as loading control. The experiment was repeated three times and a representative experiment is shown. *P < 0.05 versus wild‐type control, **P < 0.01 versus wild‐type control. (C) The ubiquitination profile of HIF‐1 was examined by using heart extracts from each group. The extracts were used for immunoprecipitation with anti‐ubiquitin antibody followed by Western blot with an antibody against HIF‐1α. (D) The interaction between HIF‐1α and SUMO‐1 was determined by immunoprecipitations using anti− HIF‐1α antibody followed by Western blotting with anti‐SUMO‐1 antibody. Western blot with anti− HIF‐1α antibody served as loading control. (E) Western blot was performed as in (A) to evaluate expression of SUMO‐1. GAPDH was used as loading control. (F) Expression of pyruvate dehydrogenase kinase‐1 (PDK‐1) was evaluated by Western blot using antibody against PDK‐1. The representative figure shows increase in PDK‐1 expression in extracts from CLP‐1 +/− mice hearts subjected to stress. GAPDH expression was used as loading control.

Mentions:
(A) Schematic representation of the experimental protocol used for the evaluation of haemodynamic parameters and infarct size. (B) A diagram of the experimental protocols for stress application and analysis of wild‐type and CLP‐1 +/− hearts (see Figs. 4 and 5).

Bottom Line:
There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts.However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically.Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

Affiliation:
Center for Cardiovascular and Muscle Research, Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 112031, USA.

ABSTRACTIn order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP-1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP-1 +/- mice hearts. Using the isolated heart model, we observed that the CLP-1 +/- hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP-1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild-type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts. However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK-1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.